Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice

Abstract

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained.

Evaluation of the appropriateness of using glucometers for measuring the blood glucose levels in mice

“…On the other hand, various methods of anesthesia are reported to have an impact on the blood glucose levels in rodents 5 . Retro-orbital sinus blood from male C57BL/6J mice reportedly showed lower elevations of the plasma corticosterone levels and lesser body weight loss as compared to blood samples obtained by facial vein phlebotomy 16 . However, retro-orbital sinus puncture may exert considerable impact on the animal welfare, by potentially causing subcutaneous hematomas and extensive tissue trauma 16 , which should be considered whenever blood samples are obtained.…”

“…Retro-orbital sinus blood from male C57BL/6J mice reportedly showed lower elevations of the plasma corticosterone levels and lesser body weight loss as compared to blood samples obtained by facial vein phlebotomy 16 . However, retro-orbital sinus puncture may exert considerable impact on the animal welfare, by potentially causing subcutaneous hematomas and extensive tissue trauma 16 , which should be considered whenever blood samples are obtained. For measurement of the blood glucose levels, the blood sampling should be minimally invasive and have minimal impact on the stress reaction of the animals, because the release of stress hormones can cause elevation of the blood glucose levels.…”

“…Retro-orbital sinus puncture in laboratory rodents is a commonly used technique for blood sampling, although its appropriateness still remains controversial 14 15 16 . This method may cause distress and injury when implemented inadequately 14 17 .…”

Applicability of Commercially Available ELISA Kits for the Quantification of Faecal Immunoreactive Corticosterone Metabolites in Mice

“…However, a blood sample gives only the momentary concentration and, therefore, merely reflects the stress response at that very instant. The blood sampling procedure itself is also problematic, since it is invasive and requires certain form of restraint, which causes stress and trauma to the animal (4,5). To circumvent these issues, faeces can be collected from the mouse cage and faecal CORT metabolites (FCM) can be extracted.…”

“…Many studies, among them certain performed in our laboratory, have applied commercially available enzymelinked immunosorbent assays (ELISAs) for the quantification of FCM for stress assessment in several animal species, including laboratory mice (3)(4)(5)(9)(10)(11)(12). However, such assays are generally designed for measuring serum CORT in its native form, with the content of native CORT being very low in faecal samples (13).…”